Abstract Staphylococcus aureus (SA) is a major human pathogen responsible for numerous chronic and relapsing infections. These infections often do not respond to treatment, leading to 12,000 deaths and $9.5 billion in health care cost annually in the US alone. Paradoxically, during in vitro susceptibility testing, isolates from these infections frequently exhibit full sensitivity to administered antibiotics, suggesting that environmental factors present in the host may influence SA antibiotic susceptibility. Understanding how these factors control antibiotic susceptibility will improve the resolution of recalcitrant SA infection, and slow the evolution of resistance. We have shown that during co-infection, interaction with Pseudomonas aeruginosa (PA) alters SA antibiotic susceptibility through 3 distinct mechanisms. PA LasA endopeptidase potentiates vancomycin- mediated lysis of SA, rhamnolipids (RLs) facilitate tobramycin uptake and killing of SA, and HQNO induces multidrug tolerance in SA. Using a panel of clinical isolates we further demonstrated that the ability of PA to antagonize or potentiate antibiotic efficacy against SA is variable and dependent on PA production of these three antistaphylococcal exoproducts. Selective pressures during chronic infection of the CF lung drive PA adaptive evolution and result in common phenotypic characteristics among late-stage CF isolates with altered production of LasA, HQNO and RLs. However, the overall effect of these changes on SA antibiotic susceptibility during co-infection is unknown. I hypothesize that mutations that occur during PA adaptation to the CF lung alter SA antibiotic susceptibility during co-infection. Further, I hypothesize that membrane permeabilization by PA RLs can be exploited to kill SA and clear chronic infection. In AIM1 I will determine how long-term adaptation to the CF lung alters PA influence on SA antibiotic susceptibility. I will use whole-genome sequencing to identify genetic hallmarks of late-stage (chronic) PA isolates that strongly potentiate or antagonize antibiotic activity against SA, relative to early-stage (acute) and reference strains. I will then introduce these and other common mutations associated with chronic PA CF infection in acute isolates and test for loss antibiotic potentiation or antagonism against SA. In AIM2 I will expand on our finding that PA RLs facilitate tobramycin-mediated eradication of SA to determine if targeting cell membrane permeability represents a viable treatment strategy against SA. We will identify other cell membrane-acting agents (CMAs) capable of potentiating aminoglycoside activity, examine the capacity of RLs/CMAs to re-sensitize tobramycin-tolerant/resistant populations, and examine the feasibility of using aminoglycosides and CMAs as a combinational therapy against SA using tissue culture and a mouse model of SA burn wound infection. In all, we expect that our findings will help improve our understanding of SA antibiotic susceptibility, and elucidate how such knowledge can be exploited to resolve currently unresolvable infections. !